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Received: 9 March 2018 Accepted: 4 May 2018 Published: xx xx xxxx
Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm Courtney Davis-Vogel 1,2, Angel Ortiz1, Lisa Procyk1, Jonathan Robeson1, Adane Kassa1, Yiwei Wang1, Emily Huang1, Carl Walker1, Amit Sethi1, Mark E. Nelson1 & Dipali G. Sashital2 Western corn rootworm (Diabrotica virgifera virgifera) is a serious agricultural pest known for its high adaptability to various management strategies, giving rise to a continual need for new control options. Transgenic maize expressing insecticidal RNAs represents a novel mode of action for rootworm management that is dependent on the RNA interference (RNAi) pathways of the insect for efficacy. Preliminary evidence suggests that western corn rootworm could develop broad resistance to all insecticidal RNAs through changes in RNAi pathway genes; however, the likelihood of field-evolved resistance occurring through this mechanism remains unclear. In the current study, eight key genes involved in facilitating interference in the microRNA and small interfering RNA pathways were targeted for knockdown in order to evaluate impact on fitness of western corn rootworm. These genes include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2. Depletion of targeted transcripts in rootworm larvae led to changes in microRNA expression, decreased ability to pupate, reduced adult beetle emergence, and diminished reproductive capacity. The observed effects do not support evolution of resistance through changes in expression of these eight genes due to reduced insect fitness. RNA interference (RNAi) is a biological process conserved across plants and animals wherein gene expression is controlled through a mechanism mediated by small 20–30 nucleotide complementary single-stranded RNAs1. The microRNA (miRNA) and small interfering RNA (siRNA) pathways provide endogenous control of gene expression, mobile genetic elements, and invading viruses2–4. Steps required for the interference response in various cellular contexts have been elucidated through study of model organisms, resulting in an advanced understanding of the RNAi pathways in the dipteran insect Drosophila melanogaster. The D. melanogaster miRNA pathway begins with the cleavage of endogenously expressed single-stranded primary miRNAs into precursor miRNAs by the microprocessor complex composed of Drosha and Pasha5,6. Precursor miRNAs are exported from the nucleus and further processed into short double-stranded RNA (dsRNA) duplexes by Dicer-1 (DCR-1) and Loquacious (LOQS)7–9. The D. melanogaster siRNA pathway is activated by long dsRNAs which can be taken up from the environment and processed into short duplexes in the cytoplasm by Dicer-2 (DCR-2) and R2D210,11. One strand of either a miRNA or siRNA duplex is loaded into Argonaute 1 (AGO1) or Argonaute 2 (AGO2), respectively, forming active RNA induced silencing complexes (RISCs)12–16. Both types of RISC bind RNAs with some degree of complementarity to the guiding small RNA, resulting in repression or cleavage of target RNA2,3. These eight proteins—Drosha, Pasha, LOQS, DCR-1, DCR-2, R2D2, AGO1, and AGO2—are the core RNAi machinery of the mi- and siRNA pathways, so designated due to their central involvement in facilitating the interference response. Both the mi- and siRNA pathways may be exploited to cause deliberate knockdown of essential genes in receptive organisms, a technique known as environmental RNAi (eRNAi) that has garnered interest in the agricultural sector as an innovative means of insect control17. Plant-produced RNAs have been demonstrated to provide protection against agricultural pests of several different phylogenetic orders18–20. The western corn rootworm (WCR – Diabrotica virgifera virgifera) is one of the costliest pests in North America, with an estimated $1.17 billion expended annually in management inputs and yield loss21. It was also the first major agricultural pest shown to be controllable through an RNAi-based transgenic trait20, and is the target of the first such trait registered by the 1
Research and Development, DuPont Pioneer, 7300 NW 62nd Ave., Johnston, IA, USA. 2Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, 2437 Pammel Dr., Ames, IA, USA. Angel Ortiz, Lisa Procyk, Jonathan Robeson, Yiwei Wang, Emily Huang and Carl Walker contributed equally to this work. Correspondence and requests for materials should be addressed to C.D.-V. (email:
[email protected])
Scientific RePOrTS | (2018) 8:7858 | DOI:10.1038/s41598-018-26129-6
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www.nature.com/scientificreports/ United States Environmental Protection Agency22. Development of transgenic traits controlling WCR through novel modes of action such as RNAi are valuable due to the pest’s history of overcoming certain chemical and biological insecticides, as well as management practices such as crop rotation23. This adaptability must be taken into consideration prior to the deployment of new control technologies so as to maximize trait efficacy and lifespan through appropriate resistance management. Development of WCR resistance to insecticidal RNAs has been speculated to be possible through upregulation of the target gene or target site insensitivity, though of particular concern would be the development of a resistance mechanism conferring protection across many or all insecticidal RNAs23,24. Such a mechanism would have to involve processes central to the RNAi response in insects. Artificially reducing expression of dcr-2 and ago2 has previously been reported to both confer complete protection to WCR adults against an insecticidal dsRNA and show no phenotypic effects in adults or larvae25–27. It was additionally shown that knockdown of drosha and dcr-1 did not seem to affect WCR adults, though effects were observed with ago1 knockdown25–27. Moreover, the siRNA pathway genes have been reported as among the 3% fastest evolving genes in D. melanogaster28, and eukaryotic organisms in general show high diversity in the presence, number, and function of RNAi pathway components29. Collectively, these reports imply that changes in certain core RNAi machinery may be sufficient to cause resistance to RNAi-based control in WCR. Recent evidence suggests evolution of RNAi pathway gene functionality in insects is a slow and complex process30–33. While certain regions of the sequences themselves may show rapid change, conserved regions that preserve protein function—and indeed the miRNA pathway genes themselves which participate in certain aspects of siRNA-mediated RNAi—show little to no evidence of positive selective pressure28. Despite hundreds of millions of years of host-pathogen interaction forcing adaptation in these sequences, including direct targeting by viral suppressors of RNA silencing, insects still rely heavily upon the RNAi pathway proteins for defense against entomopathogenic viruses31,32,34,35. Therefore, changes in expression of these genes was considered to be a more viable route to resistance than outright loss or functional mutation. The current study explores the effects of reduced expression of core RNAi pathway genes to determine whether this is a potential route to resistance. Laboratory-based probes into broad theoretical resistance mechanisms can overlook potential repercussions of such mechanisms on practical viability of the insect. Many genes participating in the function and efficiency of insect RNAi pathways are involved in multiple processes, and their alteration may have widespread consequences—especially in a natural setting35–37. Downregulation of core components of the WCR RNAi pathway is potentially one of the most direct paths to resistance against RNAi-based control suggested to date. The current study provides an in-depth assessment of the impact on WCR of lowered core RNAi machinery expression. Knockdown targets include all eight genes serving core processing roles in the mi- and siRNA pathways, due to interest in utilization of both pathways for insect control, as well as their known or suspected pathway cross-functionality in D. melanogaster7,38–44. In agreement with previous reports, no phenotypic abnormalities were observed in larval stages upon treatment with dsRNA against these targets. However, knockdown occurring in older WCR larvae resulted in decreased ability to pupate, reduced adult emergence, and diminished reproductive capacity. Additionally, decreased expression of the core RNAi machinery caused changes in expression of miRNAs. The effects on post-larval WCR observed within this study argue against changes in expression of the core RNAi machinery directing field-evolved resistance.
Results
Design of WCR RNAi machinery knockdown experiments. Double-stranded RNAs against each of the eight core RNAi machinery genes were prepared for oral administration to WCR larvae in two different types of bioassays (Fig. 1). To the extent possible, dsRNAs were designed to target all known isoforms of each gene45. The first type of bioassay utilized larval WCR early in the first instar. Approximately 24 hours post-hatch, larvae were presented with fresh diet containing either buffer, Escherichia coli β-glucuronidase (gus) dsRNA, dvssj1 insecticidal dsRNA46, or target gene dsRNAs and allowed to feed for either two or seven days without diet refresh. At the end of each time point, larvae were collected for gene expression analysis when control larvae were latefirst or late-second instar, respectively. At the end of the bioassay, larval growth and development was assessed prior to collection. Evaluation of the effects of each of the eight dsRNAs occurred over the course of several experiments, but may be directly compared. The second type of bioassay utilized larval WCR late into the third instar. Approximately two to four days prior to pupation, actively feeding larvae were exposed for 24 hours to fresh diet containing each treatment. Treated larvae were then allowed to pupate and monitored for their ability to develop into reproductively capable adults. Insects were collected for gene expression analysis when control insects were late-third-instar larvae, pupae, adult males and females prior to and following a defined egg-laying period, and eggs approximately one day from hatch. Adult emergence, mortality, egg production, and egg hatch rate was measured. Due to assay complexity, exposure to the eight dsRNAs was split into two experiments. The first experiment was a pilot study with only a water and dcr-1 dsRNA treatment, to ensure effects could be observed. The second experiment included additional negative controls and the remaining dsRNAs. Results should only be compared within an experiment due to variation in performance of the source WCR colony. Success and persistence of RNAi machinery knockdown. Effectiveness of the dsRNAs in suppressing
each target gene was evaluated using reverse transcription polymerase chain reaction (RT-qPCR). Expression analysis was conducted on larvae collected from each treatment at both two and seven days post-exposure in early-first-instar bioassays, as well as at three days post-exposure in late-third-instar bioassays (Fig. 2). A decrease in transcript levels of each target gene is observed shortly after exposure for both first and third instar larvae, consistent with the robust response of WCR to eRNAi. Some targets show increased suppression after an additional five days in early-first-instar bioassays, while others show little difference in knockdown between two and seven
Scientific RePOrTS | (2018) 8:7858 | DOI:10.1038/s41598-018-26129-6
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Figure 1. Bioassays used for knockdown of core RNAi machinery in relation to WCR life cycle. Depicted are eight points throughout the WCR life cycle relevant to the two bioassays used for exposure to dsRNA targeting core RNAi machinery: first through third larval instar, pupa, pre-reproductive adult male and female, reproductive adult male and female, young egg (
